Methods for testing for caloric restriction (CR) mimetics

ABSTRACT

Methods for treating neurological diseases and for testing Caloric Restriction (CR) mimetics or CR mimetic candidates. In one exemplary method, a CR mimetic candidate is administered to a transgenic animal and the effects of the administering are determined; the transgenic animal includes an added gene from another type of animal or a modified gene which is designed to produce a disease or ailment of another type of animal, and the method seeks to determine whether the CR mimetic candidate improves the disease or ailment. Methods relating to neurological disease and other methods relating to CR mimetic testing are also described.

CROSS REFERENCE TO RELATED APPLICATIONS

This patent application is a continuation of application Ser. No.13/154,035 filed on Jun. 6, 2011, which application is a divisional ofapplication Ser. No. 11/377,986 filed on Mar. 17, 2006, now U.S. Pat.No. 7,960,605, entitled “Methods for Testing for Caloric Restriction(CR) Mimetics.”

BACKGROUND OF THE INVENTION

1. Field

The present disclosure relates to the fields of medical and biologicalresearch and methods for discovering medical therapeutic treatments andmethods for treating certain diseases and/or ailments. Certain aspectsof this disclosure relate to properties of and uses of grape extract andsubstances related to grape extract.

2. Background

Grape extract has been studied for its use as a possible dietarysupplement and has been available commercially from a variety ofsources. For example, a form of grape extract called Regrapex-R isavailable from Interpharma Praha, a.s., of the Czech Republic.Regrapex-R is a whole extract from red grapes of Vitis vinifera, grownin southern France, and contains a natural resveratrol complex derivedfrom dried roots of Polygonum cuspidatum. Other forms of grape extractinclude grape seed extracts which are commercially available.

Examples of uses of grape extract are shown in U.S. Pat. Nos. 6,470,894and 6,638,545. U.S. Pat. No. 6,470,894 describes a composition forinclusion within a cigarette, cigar, pipe or smokeless tobacco, and thiscomposition is an antioxidant complex which is capable of scavenging andneutralizing the free radicals emanating from the burning or heatedtobacco. This composition includes glutathione, green tea and/or grapeseed extract. U.S. Pat. No. 6,638,545 describes the use of grape extractas a food complement to treat obesity and to treat, for cosmeticpurposes, cellulite. The grape extract is obtained from grape seeds orgrape marc. Other uses of grape extract are described in published U.S.Patent Applications 2005/0100617 and 2004/0047896.

The grape extract “Regrapex-R” includes resveratrol which has beenthought to be a possible caloric restriction (CR) mimetic. CR is atechnique in which subjects, such as mice or humans, are fed a diet (“aCR diet”) which has less than a “normal” amount of food for at least acertain period of time. Typically, the amount of food given in the CRdiet is less than about 50% or 40% of the minimum recommended dailyamount measured in calories. The effects of a CR diet have beenextensively studied, and numerous researchers have developed techniquesto screen for interventions, such as therapeutic substances orcompounds, which when administered to a subject on a normal diet tend tocause the subject's physiological or biological state to mimic the stateof an organism on a CR diet. Such interventions are referred to as CRmimetics because they cause the biological state of the organismreceiving the intervention to mimic the state of a similar organism on aCR diet even if the organism is not on a CR diet. The effects of CRdiets and techniques for screening for CR mimetics are described in thefollowing patents and published applications which are herebyincorporated herein by reference: U.S. Pat. Nos. 6,406,853 and 6,569,624and U.S. published applications 2005/0266438, 2005/0013776,2004/0191775, 2004/0180003, 2003/0224360 and 2003/0124540.

SUMMARY OF THE DESCRIPTION

Methods for testing CR mimetics and potential CR mimetics (e.g.“candidate CR mimetics”) are described. The methods may be employed aspart of a battery of tests (e.g. a set of tests) to screen for potentialCR mimetics and to characterize the properties of CR mimetics andpotential CR mimetics. This battery of tests may include testing forantioxidant capabilities of a potential CR mimetic and/or testing theeffect of administering the potential CR mimetic to a transgenic animal.Another aspect of the present disclosure relates to the use of grapeextract to treat neurological disorders, diseases and/or ailments suchas Parkinson's disease in humans.

In one exemplary method described herein, a method for testing a CRmimetic or CR mimetic candidate includes administering the CR mimetic orCR mimetic candidate to a transgenic animal and determining the effectsof the administering. This testing may be performed for the purpose ofdetermining whether the CR mimetic candidate is in fact a CR mimetic ormay be performed to determine the characteristics or properties of theCR mimetic candidate. The transgenic animal typically includes an addedgene from another type (e.g. species) of animal or a modified gene whichis designed to produce a disease or ailment of the another type ofanimal in the transgenic animal. The effects of the administering mayinclude an improvement in a symptom of the disease. In a specificexample provided below, administering of grape extract to a transgenicDrosophila Parkinson's disease (PD) model was shown to improve thesymptoms of PD in the male Drosophila receiving the grape extract; inparticular, grape extract given to male Drosphila having the PD gene wasshown to protect against a decline in climbing rate with age. Thisadministering may be part of a set of screening tests which are designedto determine whether a CR mimetic candidate is a CR mimetic; forexample, another screening test may include comparing levels ofbiological parameters of known CR markers to measurements of the knownCR markers from the subject having been administered the CR mimeticcandidate. In the specific example provided below, grape extract may betested as a CR mimetic by obtaining measurements of gene expressionlevels (e.g. RNA transcript levels) of known CR markers in subjectsreceiving the grape extract and comparing those measurements to knowngene expression levels, induced by a CR diet, of the known CR markers.Known CR markers include genes known to be affected (e.g. affected ingene expression levels) by a CR diet or a known CR mimetic or geneproducts of such genes or metabolites involved in biochemical pathwayswhich use those gene products.

Another aspect of the present disclosure relates to testing of theantioxidant properties of a CR mimetic or potential CR mimetic. Theantioxidant properties of the CR mimetic or potential CR mimetic may becompared to known antioxidants, and this testing may be part of a set oftests designed to determine whether a potential CR mimetic is a CRmimetic (or to characterize the CR mimetic); for example, the potentialCR mimetic, which has been or will be tested for antioxidantcapabilities, may be tested using known CR mimetic screening techniques,such as those described in U.S. Pat. Nos. 6,406,853 and 6,569,624 and inU.S. published applications 2005/0266438, 2005/0013776, 2004/0191775,2004/0180003, 2003/0224360, and 2003/0124540, to determine whether thepotential CR mimetic is a CR mimetic.

BRIEF DESCRIPTION OF THE DRAWINGS

The present disclosure is illustrated by way of example and notlimitation in the figures of the accompanying drawings in which likereferences indicate similar elements.

FIG. 1 is a flowchart showing an exemplary method of using a transgenicanimal for tests of a CR mimetic or CR mimetic candidate.

FIG. 2 is a flowchart showing an exemplary method of testing a known CRmimetic or a CR mimetic candidate for antioxidant capabilities.

FIG. 3 is a flowchart showing an exemplary method for treating aneurological disease or ailment.

FIG. 4A shows an electron spin resonance (ESR) spectra of hydroxylradicals which shows the free radical scavengering capabilities of aform of grape extract on hydroxyl radicals.

FIG. 4B is a graph showing the free radical scavengering capabilities,on hydroxyl radicals, of a form of grape extract.

FIG. 5A shows an ESR spectra which shows the free radical scavengeringcapabilities of a form of grape extract on superoxide radicals.

FIG. 5B is a graph showing the free radical scavengering capabilities,on superoxide radicals, of a form of grape extract.

FIG. 6A shows an ESR spectra which shows the free radical scavengeringcapabilities of a form of grape extract on lipid radicals.

FIG. 6B is a graph showing the free radical scavengering capabilities,on lipid radicals of a form of grape extract.

FIG. 7 is a table which compares the free radical scavengingcapabilities of a form of grape extract, labeled as “LEF,” with otherwell known scavengers, including a combination of vitamins C and E(which is labeled as “EPCK1”).

FIGS. 8A and 8B show the effect of a form of grape extract onacrolein-induced decrease in mitochondrial complex II activity in liverand brain mitochondria respectively.

FIGS. 9A and 9B show the effect of a form of grape extract on AAPH (aradical initiator that with increasing concentration represses state 3respiration) induced decrease in mitochondrial complex I activity inliver and brain mitochondria respectively.

FIG. 10A is a graph which shows the effect of a form of grape extract onthe climbing ability of male transgenic Drosophila that have the PDgene.

FIG. 10B is a chart which shows a statistical analysis of the data fromthe climbing assay shown in FIG. 10A.

FIG. 11A is a graph which shows the effect of a form of grape extract onthe climbing ability of female transgenic Drosophila that have the PDgene.

FIG. 11B is a chart which shows a statistical analysis of the data fromthe climbing assay shown in FIG. 11A.

FIG. 12A is a graph that shows the effect of a form of grape extract onlifespan for male Drosophila.

FIG. 12B is a graph that shows the effect of a form of grape extract onlifespan for female Drosophila.

DETAILED DESCRIPTION OF THE INVENTION

The various embodiments of the present inventions will be described withreference to numerous details set forth below, and the accompanyingdrawings will illustrate these various embodiments. The followingdescription and drawings are illustrative of the invention and are notto be construed as limiting the invention. Numerous specific details aredescribed to provide a thorough understanding of the present inventions.However, in certain instances, well known or conventional details arenot described in order to not unnecessarily obscure the presentinventions in detail.

Exemplary embodiments are described with reference to specificconfiguration and techniques. Certain embodiments of the presentinvention pertain to methods of screening for CR mimetics andreproducing the effects induced by a CR diet without requiring thesubject to endure an actual CR diet. Methods of identifying compoundsthat reproduce the effects induced by CR, identifying compounds thatprevent or delay the onset of age related diseases or extend longevity,and extending longevity in mammals are disclosed.

Currently, CR when started either early in life or in middle-age,represents the best established paradigm of aging retardation inmammals. See for example, Weindruch, et. al., The Retardation of Agingand Disease by Dietary Restriction, C. C. Thomas, Springfield, Ill.,1988. The effects of CR on age-related parameters are broad. CRincreases maximum lifespan, reduces and delays the onset of age relateddiseases, reduces and delays spontaneous and induced carcinogenesis,suppresses autoimmunity and general decline of immune functionassociated with aging, and reduces the incidence of several age-induceddiseases (Weindruch, supra, 1988). For example, CR delays the onsetkidney disease, cancer, autoimmune disease, and diabetes. CR reducesneuronal loss with age in mouse models of neurodegenerative disorders,including Parkinson's disease and Alzheimer's disease. CR also preventsdecline in learning ability with age as measured by psychomotor andspatial memory tasks. CR also prevents dendritic spine loss and enhancesthe brain's plasticity and repair.

Even though CR brings many beneficial effects to animals and humans, itis not likely that many will avail themselves of a CR lifestyle. As isknown, it is difficult for any animal or human to maintain a dietprogram similar to a CR diet program. There is thus a need to identify,evaluate, and develop CR mimetic compounds or drugs that are capable ofmimicking at least some of the anti-aging, anti-disease effects, andother beneficial effects of CR without the reduction of dietary calorieintake as required by CR diet programs. Furthermore, it is an aspect ofat least certain embodiments of the invention to use a battery of tests(e.g. a set of tests) which are used to search for and screen for a CRmimetic from a pool of CR mimetic candidates. This battery of tests mayinclude the conventional testing of gene expression levels or othertechniques for screening for CR mimetics (e.g., as described in U.S.Pat. Nos. 6,406,853 and 6,569,624 and U.S. published applications2005/0266438, 2005/0013776, 2004/0191775, 2004/0180003, 2003/0224360 and2003/0124540) with other tests such as tests for antioxidantcapabilities, and/or test on effects of mitochondria, and/or tests ofthe effects of administering a CR mimetic candidate to a transgenicanimal. For example, a transgenic animal which is created to have agenetically designed model of an aging disease, such as Parkinson'sdisease or Alzheimer's disease or other disease(s) commonly associatedwith age (e.g., certain types of diabetes or cancers), may be used in atest which studies the effects of administering a CR mimetic candidateto such a transgenic animal.

FIG. 1 shows an example of a method, according to one exemplaryembodiment of the present inventions, which uses a transgenic organism(such as transgenic animal or plant or other life forms) in a study of aknown CR mimetic or a CR mimetic candidate (e.g., a study to screen fora CR mimetic candidate). Operation 101, in FIG. 1, involves theselection of a type of a transgenic animal for use in the CR mimeticstudy. The transgenic animal typically includes an added gene fromanother type of animal or a modified gene which is designed to produce adisease or ailment of another type of animal in the transgenic animal.In the specific example described herein, Parkinson's disease (PD) modelDrosophila were selected for use with a CR mimetic candidate, a form ofgrape extract. Further details regarding the Parkinson's disease modelDrosophila and the particular form of grape extract are provided below.In operation 103, a known CR mimetic or a CR mimetic candidate isadministered to the selected transgenic animal (e.g. a Parkinson'sdisease model Drosophila). Then in operation 105, the effects of theadministering of the known CR mimetic or a CR mimetic or a CR mimeticcandidate to the transgenic animal are determined. FIGS. 10A, 10B, 11A,and 11B show the results of determining the effects of suchadministering of a form of grape extract to a Parkinson's disease modelDrosophila. It appears, from these specific results, that grape extractis effective in reducing the effects of Parkinson's disease, at least inthe male portion of a population. These results extend to humans becausethe particular Parkinson's disease model Drosophila were designed as atransgenic animal to model human Parkinson's disease, and thus theseresults show that grape extract may be used to treat, in humans,neurological diseases, such as Parkinson's disease. Operation 107 is anoptional operation which performs other tests using the CR mimeticcandidate to verify whether the candidate is a CR mimetic. Theseadditional tests may include conventional screening for CR mimeticcandidates such as assaying of biological parameters, such as geneexpression levels (e.g., RNA transcript levels), of certain CR markers,and the comparing of those measured gene expression levels to measuredgene expression levels of those CR markers of subjects on a CR diet or aknown CR mimetic. See for example, U.S. Pat. Nos. 6,406,853 and6,569,624 and U.S. published applications 2005/0266438, 2005/0013776,2004/0191775, 2004/0180003, 2003/0224360 and 2003/0124540. CR markersinclude genes (and hence expressed gene levels as measured byconcentrations of RNA transcripts) which are known to be affected by aCR diet (based on the studies of the differential effect of CR dietsrelative to normal diets) or by a known CR mimetic or gene products(e.g. proteins) of such genes or metabolites involved in biochemicalpathways which use those gene products.

FIG. 2 shows an example of a method, according to another exemplaryembodiment of the present inventions, which studies the antioxidantcapabilities of a known CR mimetic or a CR mimetic candidate. FIGS. 4A,4B, 5A, 5B, 6A, 6B and 7 show specific examples, which are discussedfurther below of studies which determine antioxidant capabilities of aCR mimetic candidate. Alternative methods may study the protectiveeffects of a CR mimetic candidate on mitochondrial functions, andspecific examples of these alternative methods are shown in FIGS. 8A,8B, 9A and 9B. Referring to FIG. 2, operation 201 selects a known CRmimetic or a potential CR mimetic (“CR mimetic candidate”), and inoperation 203, the antioxidant capabilities of the selected CR mimeticor CR mimetic candidate are determined. Then in optional operation 205,the determined antioxidant capabilities are compared to knownantioxidants; FIG. 7 shows an example of such a comparison. The methodshown in FIG. 2 may also optionally include operation 207, in whichother tests are performed to determine whether the CR mimetic candidatetested in operation 203 appears to be a CR mimetic. In other words,operation 207 performs other tests, such as conventional CR mimeticscreening tests (e.g. as described in U.S. Pat. Nos. 6,406,853 and6,569,624) or the testing of operations 101, 103, and 105 of FIG. 1 orthe lifespan testing shown in FIGS. 12A and 12B. These tests, ifperformed, form a set of tests for testing and screening a CR mimeticcandidate as part of a process of identifying a CR mimetic from a poolof CR mimetic candidates.

FIG. 3 relates to another aspect of the present inventions. This aspectinvolves the treatment of neurological diseases or ailments, such asParkinson's disease or Alzheimer's disease. The particular method shownin FIG. 3 has been used to treat Parkinson's disease model Drosophila asis shown in FIGS. 10A, 10B, 11A, and 11B. Operation 301 of FIG. 3involves determining that a particular subject (e.g. a human patient)has a neurological disease or ailment (e.g. the human patient hasParkinson's disease). A therapeutically effective amount of grapeextract (e.g. Regrapex-R) is then administered to the subject (e.g. thehuman patient who has Parkinson's disease). The amount may be determinedbased on its effect by monitoring the effect in operation 305. In otherwords, the amount may be varied to determine whether a current amounthas a beneficial effect and if not the current amount can be increasedor decreased. It is anticipated that effects will be observed for anaverage subject when the ratio between the mass of food consumed in oneday and the mass of grape extract (in the form of Regrapex-R) given inone day is about 2000 to 1 (or about 1-2 g per day for an average adultweighing between 120-200 lbs.). More details will now be provided forthe PD Drosophila model and the tests which produced the results shownin FIGS. 10A, 10B, 11A, 11B, 12A and 12B.

The form of grape extract which was used to produce the resultsdescribed herein is a commercially available form of grape extract knownas “Regrapex-R” which is available from Interpharma Praha, a.s. of theCzech Republic. Regrapex-R is described on Interpharma's website(http://www.interpharma-praha.com). Regrapex-R is a whole grape (vitisvinifera) extract enriched with purified powdered extract, containingresveratrol, from dried roots of Polygonum cuspidatum; one gram of bulkRegrapex-R consists of about 800 mg whole grape extract and 200 mg ofdried root powder from Polygonum cuspidatum. This product containsconcentrated active principles found in red grapes (simple polyphenols,flavonoids, anthocyanins and OPC's).

The Drosophila studies shown in FIGS. 10A, 10B, 11A, 11B, 12A and 12Buse a transgenic Drosophila having been genetically engineered to havecertain added genes which produce a phenotype which resemblesParkinson's disease in humans. This type of Drosophila is referred to asa Parkinson's disease (PD) Drosophila model. Transgenic alpha-synucleinDrosophila were obtained from Dr. Feany at Harvard University (Feany andBender, 2000). The Parkinson's disease model flies express a human formof alpha-synuclein and produce adult-onset loss of dopaminergic neurons,filamentous intraneuronal inclusions containing alpha-synuclein andlocomotor dysfunction. Since this Drosophila model recapitulates theessential features of the human disorder, it makes possible a powerfulgenetic approach to study Parkinson's disease in humans.

The control non-PD flies (UAS—wild-type alpha-synuclein/+) and PD flies(Ddc-GAL4/+; UAS—wild-type alpha-synuclein/+) were maintained at 25° C.on a 12-h light/dark cycle in bottles containing an agar, corn meal,sucrose, water, and dried yeast medium (Pendleton et al., 2002).Propionic acid was added to prevent fungal growth. Drugs (Regrapex-R)were added to the medium at the final concentrations marked in theresults (e.g. the grape extract, labeled “LEF,” dose is mg/100 g culturemedium, so “LEF-1.25” in FIGS. 10A-12B means 1.25 mg of Regrapex-R per100 g of culture medium) and the mixture was heated to a boil withcontinuous stirring. UAS—wild-type alpha-synuclein flies were used asthe basis for comparison with the PD flies because they are femaleparent of PD flies and a genetically steady, constant breeding stockwhose functional behavior in our standard locomotor assay describedbelow was identical with the transgenic stock during the first 6 or 12days after eclosion.

Flasks containing the desired stocks were emptied leaving pupa to emerge(eclose) as adults. Newly eclosed flies of both the non-PD control andPD strains were placed in culture tubes (10 flies per tube) thatcontained drug-treated food which are renewed every 3 days. Controlorganisms were treated similarly except that the medium was drug free.The following day, the number of dead flies is recorded at 3-dayintervals. At 6-day intervals thereafter, the adults were tested in theclimbing assay. Assays continued until day 48.

Climbing Assay

The climbing assay was performed as described (Feany and Bender, 2000;Pendleton et al., 2002). Ten flies were placed in an empty vitreous110×27-mm vial, around which a horizontal line 80 mm above the bottom ofthe vial was drawn. After the flies had acclimated for 10 min at roomtemperature, every group was assayed at random, to a total of 10 trialsfor each. The procedure involved gently tapping the flies down to thebottom of the vial. The number of flies above the mark of the vial wascounted after 10 s of climbing, and repeated for 10 times to get themean number of flies above the mark in the vial. These values were thenaveraged, and a group mean and standard error were obtained. Theresulting mean was used as the overall value, and the exponent ofclimbing ability was determined for each single group of flies on aparticular day. Where appropriate, the mean values of the various flygroups were statistically compared using Student's t test. All climbingassays were performed in an isolation room at 25° C., 60-70% humidityunder standard lighting conditions.

The results of the climbing assay (shown in FIGS. 10A, 10B, 11A, and11B) show that, at least for the male PD Drosophila model, theadministered grape extract shows a positive effect (e.g. delayed onsetof loss of climbing ability relative to male PD Drosophila model (“PDmale”) which did not receive the grape extract “drug”). FIG. 10A showsthe result of a climbing assay using male control Drosophila (“alpha synmale” which do not express the human alpha synuclein gene since theylack the promoter for this transgene), “PD male” Drosophila (withoutreceiving grape extract), and male PD Drosophila having received varyinglevels of grape extract (from 0.08 mg of Regrapex-R/100 g of culturemedium to 2.5 mg of Regrapex-R/100 g of culture medium). FIG. 10B showsa statistical analysis of the effect of administered grape extract onclimbing response (from the data shown in FIG. 10A) in male Drosophila.These in vivo assays show that, at least for male Drosophila, theadministered grape extract ameliorated the loss of motor functionassociated with Parkinson's Disease and an improvement in the climbingability compared to the PD male Drosophila that were not administeredthe grape extract. FIG. 11A shows the result of a climbing assay usingfemale control Drosophila (“alpha syn female”), “PD female” Drosophila(without receiving grape extract) and female PD Drosophila havingreceived varying levels of grape extract (from 0.08 mg of Regrapex-R/100g of culture medium to 2.5 mg of Regrapex-R/100 g of culture medium).FIG. 11B shows a statistical analysis of the effect of administeredgrape extract on climbing response (from the data shown in FIG. 11A) infemale Drosophila. These in vivo assays appear to show that, at leastfor female Drosophila, the administered grape extract had no beneficialeffect on the climbing ability of female PD Drosophila.

A lifespan study was also performed on the PD Drosophila lines; theresults of this study are shown in FIGS. 12A and 12B. Again, the PDDrosophila (male and female) were either given no grape extract (a PDDrosophila control) or grape extract at a certain dosage (e.g.“LEF-1.25” meaning the culture medium for PD Drosophila receiving thisdose had 1.25 mg of Regrapex-R per 100/g of culture medium). The normalDrosophila (“alpha syn male or female” transgenic Drosophila for humanalpha synuclein but lacking a promoter and thus not expressing alphasynuclein) were also included in this study. It appears that, to atleast some extent, the grape extract acts as a CR mimetic, to the extentit increases lifespan in the female PD Drosophila model.

Another aspect of the inventions related to in vitro studies of theantioxidant capabilities (shown in FIGS. 4A-7) of grape extract and theability of grape extract to protect mitochondria (shown in FIGS. 8A-9B).As noted above, these in vitro studies (of one or both types) may bepart of a battery of tests on a CR mimetic candidate, and this batterymay include tests with one or more transgenic animals (e.g. as shown inFIG. 1 and described herein) and may include conventional geneexpression level comparisons showing RNA transcript levels of selectedCR markers for a first subject on a CR diet and a second subject, of thesame species as the first subject, being administered the CR mimeticcandidate, but on a normal diet. This battery of tests may be used toscreen for CR mimetics from a pool of CR mimetic candidates.

As shown in FIGS. 4A-7, grape extract (in this case Regrapex-R)effectively scavenged hydroxyl, superoxide, and lipid free radicals. Theresults of the scavenging of hydroxyl radicals by this grape extract areshown in FIGS. 4A and 4B and the results of the scavenging of superoxideradicals by this grape extract are shown in FIGS. 5A and 5B, and theresults of the scavenging of lipid radicals by this grape extract areshown in FIGS. 6A and 6B. In all cases, the effect of this grape extracton the different types of free radicals was detected using spin trappingagents (such as DMPO) with an electron spin resonance (ESR)spectrometer. (See Liu and Mori, 1992; Liu and Mori, 1993; and Liu andMori, 1993). The grape extract was prepared in several differentconcentrations in DMSO (dimethyl sulfoxide) as shown in FIGS. 4A-6B; ineach case the amount of Regrapex-R in DMSO is in mg of Regrapex-R perml) of DMSO. FIGS. 4A and 4B show a dose dependent scavenging effect onthe hydroxyl radical with an IC₅₀ of 24.6 mg/ml. This compares favorablyto a potent hydroxyl radical scavenger made by combining vitamin C withvitamin E (referred to as EPCK1 in FIG. 7). FIGS. 5A and 5B show a dosedependent scavenging effect on superoxide radicals with an IC₅₀ 0.0026mg/ml. FIGS. 6A and 6B show a dose dependent scavenging effect on lipidradicals with an IC₅₀ of 3.68 mg/ml; this scavenging effect of grapeextract is similar to EPCK1 as shown in FIG. 7 which shows a comparisonof this form of grape extract (Regrapex-R), labeled as LEF, with avariety of known free radical scavengers, including ALC(Acetyl-L-Caritine), LA (∝-lipoic acid), EPCK1 (a diester ofalpha-tocopherol (vitamin E) and ascorbic acid (vitamin C), Resv(Resveratrol), UTR and C-Med (both formulations of “cats claw extract”).

FIGS. 8A-9B show the results of tests to investigate the ability of aform of grape extract (Regrapex-R, in this case) to protect mitochondriafrom oxidative damage. At least in the results of FIG. 9A, grape extractshowed some ability to protect mitochondria from chemically inducedoxidative damage. The mitochondria were prepared in the followingmanner: Male Sprague Dawley rats (180-200 g) were purchased fromShanghai SLAC Laboratory Animal Co. Ltd (Shanghai, China). The animalswere terminated by decapitation after an overnight fast and livers wereremoved for immediate mitochondrial isolation. Mitochondria wereisolated as described {Krahenbuhl, 1991} with slight modification.Briefly, tissues were rinsed with saline, weighed, and put into ice-coldisolation buffer containing 0.25 M sucrose, 10 mM Tris, 0.5 mM EDTA, pH7.4. Tissues were sheared carefully to mince, and rinsed to get rid ofresidual blood, and then homogenized in 2.5 vol of isolation buffer. Thehomogenate was adjusted to 8 vol with isolation buffer and centrifugedat 1,000 g for 10 min; the supernatant fraction was decanted and saved.The pellet was washed once with 2 vol of isolation buffer. Thesupernatant fractions were combined and centrifuged at 10,000 g for 10min. The mitochondrial pellet was washed twice with isolation buffer.All above operations were carried out at 4° C. The mitochondrial proteinconcentration was determined using the BCA™ Protein Assay kit (Pierce23225) using bovine serum albumin (BSA) as a standard. Freshly isolatedmitochondria were either used immediately for respiration andpermeability transition assays, or stored at −80° C. until enzymeanalysis. After isolation and preparation, the mitochondria were exposedto acrolein (in the case of the tests shown in FIGS. 8A and 8B) or toAAPH (in the case of the tests shown in FIGS. 9A and 9B). Acrolein'seffect on isolated mitochondria has been described by M. J. Picklo andT. J. Montine in “Acrolein inhibits respiration in isolated brainmitochondria.” Biochem Biophys. Acta, 2001, Feb. 14, 1535 (2), pages145-152. AAPH's effect on mitochondria has been described by T. Kanno,et al., in “Dysfunction of mouse liver mitochondria induced by2,2′-azobis-(2-amidinopropane) dihydrochloride, a radical initiator, invitro and in vivo.” Free Radical Res., 1994 Sep.; 21(4), pages 223-234.In the case of the tests shown in FIGS. 8A and 8B, grape extract showedno apparent protective effect against acrolein induced oxidative damageof both types (liver and brain) mitochondria. In the case of the testsshown in FIGS. 9A and 9B, grape extract showed (in liver mitochondriabut not brain mitochondria) a dose dependent inhibition on AAPH induceddamage at 10 and 50 μg/ml but the inhibitory effect was lost at higherconcentrations.

While particular embodiments of the inventions have been shown anddescribed, it will be appreciated that changes and modifications can bemade without departing from the inventions in their broader aspects, andtherefore the following claims are to encompass within their scope allsuch changes and modifications.

The invention claimed is:
 1. A method of testing a Caloric Restriction(CR) mimetic or a CR mimetic candidate, the method comprising: selectingat least one of the CR mimetic or the CR mimetic candidate;administering the selected at least one of the CR mimetic or the CRmimetic candidate to a subject; determining antioxidant capabilities ofthe at least one of the CR mimetic or the CR mimetic candidate, whereinthe determining includes determining: (i) a half maximal inhibitoryconcentration (IC₅₀) of the CR mimetic or the CR mimetic candidatehaving a dose dependent scavenging effect on a hydroxyl radical isconsistent with a scavenging effect of a radical scavenger made bycombining vitamin C and vitamin E, (ii) an IC₅₀ of the CR mimetic or theCR mimetic candidate having a dose dependent scavenging effect on asuperoxide radical is consistent with a scavenging effect of a knownsuperoxide radical scavenger and (iii) an IC₅₀ of the CR mimetic or theCR mimetic candidate having a dose dependent scavenging effect on alipid radical is consistent with a scavenging effect of a known lipidradical scavenger; and determining in the subject, a level of abiological parameter of a known CR marker by comparing the level of theknown CR marker to measurements of a corresponding CR marker from asubject having been administered at least one of the CR mimetic or theCR mimetic candidate, the biological parameter of the known CR markerincludes an RNA transcript level of a gene known to be effected in geneexpression by a CR diet or a known CR mimetic.
 2. A method as in claim 1further comprising: comparing the determined antioxidant capabilities toknown antioxidants.
 3. A method as in claim 1 wherein antioxidantcapabilities are determined for only the CR mimetic candidate andwherein the known antioxidants comprise at least one of vitamin C,vitamin E, Acetyl-L-Carnitine, and α-lipoic acid.
 4. A method as inclaim 3 wherein the comparing of levels of biological parameters is partof a battery of screening tests designed to identify CR mimeticcompounds and the screening tests include the determining of antioxidantcapabilities.
 5. The method as in claim 1 wherein determining thescavenging effect of the CR mimetic or the CR mimetic candidate on thehydroxyl radical, on the superoxide radical, and on the lipid radicalcomprises detecting the effect of the CR mimetic or the CR mimeticcandidate on these radicals with a spectrometer.